Morphine is one of the most commonly prescribed opioids to treat pain. However, prolonged use often leads to the development of tolerance characterized by loss of effectiveness and dose escalation. Morphine tolerance is in part due to a loss of signaling from mu opioid receptors (MOR) independent of a change in receptor levels. Despite years of study, the mechanisms causing this loss of signaling remain unsolved. Traditionally, tolerance is measured in cells or tissue removed from a chronically treated animal and washed free of morphine for several hours. Preliminary experiments have discovered that if brain slices from a morphine-treated rat are instead maintained in morphine continuously, the morphine response is significantly smaller compared to slices in which morphine was washed out, indicating that a portion of tolerance can recover within a relatively short wash. This rapid recovery following morphine removal is newly identified and may have appreciable importance to the persistence of tolerance. The goal of this proposal is to better understand morphine tolerance by carefully examining the often overlooked desensitization that is maintained by the continuous presence of morphine. The overall hypothesis is that two types of tolerance (long-lasting tolerance and chronic desensitization) develop during long-term morphine treatment distinguished by the time-course of recovery, which have different mechanisms and depend on treatment paradigm. This hypothesis will be tested using locus coeruleus neurons contained in brain slices from naove or morphine-treated animals. Morphine effects will be measured using whole-cell voltage-clamp electrophysiology to detect MOR-mediated activation of G protein-coupled inwardly rectifying potassium (GIRK) channels. The specific aims of this proposal are as follows: (1) to identify the long-lasting tolerance and chronic desensitization that results from long-term continuous morphine treatment of rats and mice, (2) to compare this to tolerance and desensitization following long-term intermittent morphine treatment and (3) to determine the role of 2-arrestin 2 in the development of long-lasting tolerance (using 2-arrestin 2 knockout mice) and the role of ser/thr phosphatases in development of and recovery from chronic desensitization (using okadaic acid). Together, this proposal will provide new information about chronic desensitization that develops after long-term morphine treatment. Focusing on morphine specifically will help identify mechanisms involved in tolerance to this most commonly used opioid agonist.